Experimental Leishmania major infection in mice has been of immense interest as it was among the first models to demonstrate the importance of the Th1/Th2 balance to infection outcome in vivo. The Th2 polarization that accompanies and promotes the development of non-healing cutaneous lesions in BALB/c mice, however, has failed to adequately explain the mechanisms underlying non-healing forms of Leishmaniasis in humans. We have studied a L. major strain from a patient with non-healing lesions that also produces progressive lesion development with ulcerations and high parasite burden in conventionally resistant C57BL/6 mice. Surprisingly, these mice develop a strong and sustained Th1 response, as evidenced by high levels of IFN-gamma production by Leishmania specific cells in the draining lymph node and in the ear lesion, and an absence of IL-4. Infection of IL-10-/- mice, blockade of the IL-10R or depletion of CD25+ cells during the chronic phase of the infection promotes parasite killing, indicating that IL-10 and regulatory T cells play a role in rendering the Th1 responses relatively ineffective at controlling infection in the inflammatory site. Mice with severe,non-healing primary lesions were nonetheless resistant to reinfection in the other ear. We suggest that non-healing infections in animal models that are explained not by aberrant Th2 development, but by overactivation of homeostatic pathways designed to control inflammation, provide better models in which to understand non-healing or reactivation forms of leishmaniasis seen in humans. The inability to reveal a Th2 polarity associated with many non-healing or systemic forms of leishmaniasis in humans has questioned the relevance of the classical TH1/Th2 paradigm to clinical disease, and has invited the identification of alternative cells and cytokines involved in susceptibility. Based on our previous studies carried out in Sudan and India, the severity of human visceral leishmaniasis was associated not with increased levels of IL-4 mRNA, but of IL-10 mRNA, detected in lesional tissue. These observations were extended by real time PCR analysis of a large series of splenic biopsies obtained from India kala-azar patients pre- and post-treatment with Amphotericin B. The data generated so far reinforce the elevated levels of IL-10 mRNA present in patients with active VL but does not support the conclusion that CD4+CD25+Foxp3+ Treg cells are the major source of IL-10. These cells are not increased in frequency in PBMC or in spleen in active VL. Importantly, the data does provide good evidence that other T cells (CD4+ and/or CD8+) are the major source of the elevated IL-10mRNA in active VL. These cells are found in both spleen and peripheral blood and their numbers seem to decline following treatment. CD4+ and CD8+ T cell responses have been shown to be critical for the development and maintenance of acquired resistance to Leishmania major. We have analyzed the potential of L. major transgenic parasites, expressing the model antigen ovalbumin (OVA), to be presented by APC to OVA specific OT-II CD4+ or OT-I CD8+ T cells. Truncated OVA was expressed in L. major as part of a secreted or non-secreted chimeric protein with L. donovani 3? nucleotidase (NT-OVA). Dendritic cells (DC) but not macrophages infected with L. major that secreted NT-OVA could prime OT-I T cells to proliferate and release IFN-g. A diminished T cell response was observed when DC were infected with parasites expressing non-secreted NT-OVA, or with heat-killed parasites. Inoculation of mice with transgenic parasites elicited the proliferation of adoptively transferred OT-I T cells and their recruitment to the site of infection in the skin. Together, these results demonstrate the possibility to target heterologous antigens to specific cellular compartments in L. major, and suggest that proteins secreted or released by L. major in infected DC are a major source of peptides for the generation of parasite-specific CD8+ T cells. The presentation of the SIINFEKL peptide by L. major NT-OVA-infected DC in vitro or by APCs involved in cross-priming in vivo, was not affected by the absence of TAP1. In contrast, cross-presentation of OVA by DC infected with Toxoplasma gondii expressing a P30-OVA fusion protein was completely TAP-dependent. When we extended these studies to the cross-presentation of wild-type L. major antigens recognized by in vivo primed CD8+ T cells, a dispensable role for TAP1 was still observed. Cross-presentation of OVA on coated latex beads could also bypass the requirement for TAP but only with high concentrations of OVA, indicating that reliance on phagosomal enzymes to generate peptide ligands is less efficient than proteosomal processing. These findings suggest that Leishmania may have evolved strategies to impair or avoid the efficient TAP-dependent, cross-presentation machinery associated with ER-mediated phagocytosis to delay CD8+ T cell priming during infection. Numerous experimental vaccines have been developed with the goal of generating long-term cell-mediated immunity against Leishmania, yet inoculation with live, wild-type L. major remains the only successful vaccine in humans. We examined the expression of immunity at the site of secondary, low dose challenge in the ear dermis to determine the kinetics of parasite clearance, and the early events associated with the protection conferred by live vaccination with L. major in C57BL/6 mice. Particular attention was given to the route of vaccination. We observed that the rapidity, strength, and durability of the memory response following live, sub-cutaneous vaccination in the footpad is even greater then previously appreciated; Ag-specific IFN-g-producing T cells infiltrate the secondary site by 1.5 weeks and viable parasites are cleared as early as 2.5 weeks following re-challenge, followed by a rapid drop in IFN-g+CD4+ cell numbers in the site. In comparison, live intra-dermal vaccination in the ear generates immunity that is delayed in effector cell recruitment to the re-challenge site, and in the clearance of parasites from the site. This compromise was associated with a rapid recruitment of IL-10 producing CD4+ T cells to the re-challenge site. Treatment with anti-IL-10-receptor or anti-CD25 antibody enhanced early parasite clearance in ear-vaccinated mice, indicating that chronic infection in the skin generates a population of regulatory cells capable of influencing the level of resistance to re-infection. A delicate balance of TEFF and TREG cells may be required to optimize the potency and durability of vaccines against Leishmaniasis and other intracellular pathogens.